KR20180126770A - Fusion protein comprising C-terminus from lamprey VLRB protein linked to hagfish VLRB protein with deleted hydrophobic tail domain and uses thereof - Google Patents

Abstract

The present invention relates to a polyvalent antibody having increased binding force to a target antibody. The polyvalent antibody comprises: a recombinant expression vector comprising polynucleotide which performs coding of a C-terminal domain of lamprey-derived VLRB protein linked to 3′-end of a gene for performing coding of hagfish-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain has been removed; a host cell transformed by the recombinant expression vector; fusion protein which is produced by the host cell and has C-terminal of lamprey VLRB protein linked to hagfish-derived VLRB protein from which a hydrophobic tail domain has been removed; and a polymer formed by self-assembly of the fusion protein. The hagfish-derived fusion antibody is expected to be advantageously applied to development of a biomarker and a diagnosis field.

Description

A fusion protein comprising a C-terminal sequence of a VLRB protein originating from the seventh eel to a VLRB protein from which a hydrophobic tail domain is removed, and a use thereof, and a fusion protein comprising the C-terminus from lamprey VLRB protein linked to hagfish VLRB protein with a deleted hydrophobic tail domain and uses thereof}

The present invention relates to a fusion protein in which the C-terminal sequence of the VLRB protein derived from the seventh eel is linked to the VLRB protein derived from the hagfish and the use thereof, wherein the hydrophobic tail domain is removed.

In general, antibodies are known to be immunoglobulin (Ig) type proteins present in most vertebrate animals. Antigen-specific binding ability of antibodies is widely used in biotechnology and medicine fields. However, antibodies have limitations such as limitations of binding area, restriction of epitope selection, and complicated production processes. As a result, many researchers are now trying to find new antibody candidates.

Recently, the existence of variable lymphocyte receptor (VLR) proteins, which play a role similar to immunoglobulins responsible for infectious diseases of poultry, has been found in hagfish and lamprey, the oldest vertebrate animals. VLR-A and VLR-C play the same role as mammalian T-lymphocytes, and VLR-B plays a role as a B-lymphocyte type secreted from lymphocyte-like cells. The VLR protein is formed by a combination of LRR (leucine-rich repeat) modules. The components include amino acid terminal (N-terminal) to SP (signal peptide), LRRNT (N-terminal capped LRR), LRRVs , CP (connecting peptide), C-terminal capped LRR (LRRCT), Stalk and HP (hydrophobic tail) domains. Among them, the LRRV module, which is an antigen recognition part, is known to include one to a maximum of nine. It has been found that VLR proteins, which are assembled by somatic rearrangement, have a theoretical diversity of about 10 ¹⁴ or more.

Many attempts have been made to replace the Ig protein after the identification of a new type of antibody, the VLR protein, in the hagfish and chilled eel. However, these attempts have been made using only the VLR protein of Chilo-long eel, which is a C-terminus domain having a very different sequence among the structures of the globular eel or the horseshoe-derived VLR protein having a generally similar structure Because. Unlike the C-terminal region of the Eagle VLR, which is highly hydrophobic by hydrophobic amino acids, there are cysteine residues and GPI (Glycosylphosphatidylinositol) cleavage sequences at the C-terminus of the globular eel VLR that induce disulfide bonds. Due to the above characteristics, the VLRs of the Seven Eel are formed by secreted 8- or 10-mer VLR complexes.

Thus, in the present invention, the C-terminal region of the hydrophobic C-terminal region is replaced with the C-terminal region of the VLRB protein derived from the Corynebine Eel. .

Korean Patent Laid-Open Publication No. 2009-0024648 discloses a 'fusion method between LRR family proteins', Korean Patent Publication No. 2016-0147787 discloses 'humanized variable lymphocyte receptor (VLR) and related compositions and uses' However, the fusion protein in which the C-terminal sequence of the VLRB protein derived from the seventh eel is linked to the VLRB protein derived from the fagopyrum, from which the hydrophobic tail domain of the present invention has been deleted, and its use have not been described.

The present invention has been made in view of the above-described needs. The present inventors have found that when the C-terminal hydrophobic tail domain is replaced with the C-terminal of the VLRB protein, while maintaining the binding site with the antigen among the VIR- Fusion proteins were prepared. As a result of transforming the host cell with a recombinant vector containing a nucleic acid encoding the fusion protein, it was confirmed that the fusion protein of the present invention was formed into a multimer of an 8- or 10-complex in the host cell, Type monoclonal antibody of the present invention has a significantly higher binding ability to the target antigen than a monoclonal (monomorphic) monoclonal antibody having the same antigen recognition site.

In order to solve the above-described problems, the present invention provides a method for coding a C-terminal domain of a VLRB protein derived from a lentil-derived Echinacea eel connected to the 3'-terminal of a gene encoding a VLRB (variable lymphocyte receptor B) protein from which a hydrophobic tail domain is removed A recombinant expression vector comprising a polynucleotide is provided.

In addition, the present invention provides a host cell transformed with said recombinant expression vector.

In addition, the present invention provides a fused protein produced by the host cell, wherein the C-terminal domain of VLRB protein derived from Echinococcus luciferase-derived VLRB protein is removed, wherein the hydrophobic tail domain is removed.

In addition, the present invention provides a multivalent antibody having an increased binding capacity to a target antigen, which is composed of a multimer of an 8-mer or 10-mer by self-assembly of the fusion protein.

The present invention also relates to a method for producing a multivalent antibody having increased binding ability to a target antigen, comprising culturing a host cell transformed with the recombinant expression vector to obtain a multimer of an 8- or 10-complex of the fusion protein .

In addition, the present invention provides a multivalent antibody with increased binding affinity to a target antigen produced by the method.

In addition, the present invention provides a method for detecting a target antigen by treating the above-mentioned polyvalent antibody with a suspected sample containing a target antigen.

In addition, the present invention provides a composition for detecting a target antigen containing the polyvalent antibody as an active ingredient.

The antibody of the present invention has 8 or 10 binding sites for an antigen rather than a simple antibody, thereby maximizing the antigen-antibody binding ability and eliminating the hydrophobic tail domain, thereby increasing the expression rate in the host cell. Stability is also increased. Unlike the mouse antibody, the horseshoe-shaped fusible antibody is composed of a single peptide and is free of genetic manipulation. Therefore, the horseshoe-derived fusing antibody of the present invention uses a high binding force and specificity for the antigen, It will be useful.

Figure 1 is a vector map of pKINGeo / ccdB and pTEL used for wild-type VLRB protein expression or VLRB fusion protein expression.
Figure 2 shows the structure (A) of the wild-type Echinocyte VLRB protein and the Western blotting result (B) of the cell culture and cell lysate of HEK 293-F cells transfected with a randomly selected wild type Echinoid VLRB protein.
FIG. 3 is a schematic diagram of the experiment (A) for replacing the hydrophobic C-terminus of the Echinochloa VLRB protein with the C-terminal sequence of the Echinochloa enteric VLRB protein and the expression pattern (B) of the western blotting confirmed under reducing conditions.
FIG. 4 shows the results (A and B) of the pTEL plasmid cloned from the VLR library derived from the horses which were immunized with VHSV for preparation of the wild-type VLRB fusion protein substituted with the C-terminal sequence of the Chilo suppressor VLRB protein, (C) results of Western blotting of the cell culture of 293-F cells transfected with the clones.
FIG. 5 shows the results of confirming the VHSV-specific binding potency of the Eagle VLRB fusion protein clone substituted with the C-terminal sequence of the Chilo suppressor VLRB protein by ELISA.
FIG. 6 is a graph showing the results (A) of the polymer formation of 43LC and 7LC selected as having a VHSV-specific binding ability among the wild-type VLRB fusion protein clones substituted with the C-terminal sequence of the Chilo suppressor VLRB protein, (B and C) and Western blotting results (D) using 43LC and 7LC as the primary antigens. 43LC or 7LC; A cell culture of 293-F cells transfected with a recombinant vector comprising a hagfish VLRB fusion protein 43 clone or a 7-clone gene substituted with the C-terminal sequence of the Chilo suppressor VLRB protein, AIV; Avian influenza virus, HA; Hemagglutinin, AgX; Antigen-free treatment, VNNV; Viral neurotrophic virus, G protein; Surface glycoprotein of VHSV.
FIG. 7 shows the results of immunocytochemical analysis of the HINAE cells treated with 43LC and 7LC for VHS virus infection.
FIG. 8 shows the results of monoclonal monoclonal antibody 43LC and 43LC monoclonal antibodies expressing only the VLRB part of the hagfish by electrophoresis under reducing and non-reducing conditions.
FIG. 9 shows the result of ELISA for the antigen-specific binding ability of monoclonal monoclonal antibody 43LC and 43LC, which express only the VLRB part of the hagfish.

In order to accomplish the object of the present invention, the present invention provides a method for producing a VLRB protein comprising the steps of: (1) isolating a C-terminal domain of a VLRB protein derived from a chloramphenicol-derived eel, which is linked to the 3'- end of a gene encoding a VLRB (variable lymphocyte receptor B) Lt; / RTI > polynucleotide encoding a recombinant expression vector.

In the recombinant expression vector of the present invention, the wild-type VLRB protein from which the hydrophobic tail domain is removed has a signal peptide (SP) at the N-terminus, a signal peptide (LRRNT (N-terminal capped LRR, leucine-rich repeat (LRR), variable LRR modules, CP (connecting peptide), LRRCT (C-terminal capped LRR), and Stalk domains. The antigen recognition sites, LRRVs and LRRCT Domain and is capable of binding to the target antigen.

In the recombinant expression vector according to an embodiment of the present invention, the VLRB protein derived from the wild boar, in which the hydrophobic tail domain is removed, may be a murine immunoglobulin κ chain leader sequence, And a signal peptide or sequence capable of further enhancing the extracellular secretory ability of the recombinant protein can be used without limitation.

In the recombinant expression vector according to an embodiment of the present invention, the murine immunoglobulin κ chain leader sequence may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the recombinant expression vector according to an embodiment of the present invention, the gene coding for the VLRB protein derived from Euphorbia, from which the hydrophobic tail domain has been removed, may be a gene coding for the VLRB protein having the best binding ability to the target antigen , Or from the VLRB cDNA library of hagfish that has been immunized with the target antigen.

In the recombinant expression vector of the present invention, the polynucleotide encoding the C-terminal domain of the VLRB protein derived from the Chilo suppressor can include a nucleotide sequence represented by the nucleotide sequence shown in SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the polynucleotide encoding the C-terminal domain of the lentil Eel VLRB protein has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 70% May comprise a nucleotide sequence having at least 95% sequence homology. &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The C-terminal domain of the lentil Eel VLRB protein contains eight cysteine (Cys) residues and a GPI (Glycosylphosphatidylinositol) cleavage sequence. Due to these characteristics, the VLRB protein of Chilo suppressor is formed into VLRB polymorph of about 400 kDa, which is composed of 8 ~ 10 monomers, and is expressed on the cell surface and secreted into the serum.

The term " recombinant " refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term " recombinant expression vector " means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

An expression vector comprising a gene coding for the horseshoe-derived VLRB protein from which the hydrophobic tail domain of the present invention has been removed, a polynucleotide encoding the C-terminal domain of the VLRB protein derived from cholesterol eel and an appropriate transcription / translation regulatory signal, Method. ≪ / RTI > Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

The recombinant expression vector may preferably comprise one or more selectable markers, but is not limited thereto. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell.

The present invention also provides a host cell transformed with said recombinant expression vector.

As a host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, any host cell known in the art may be used, and examples thereof include Escherichia coli Rosetta, Escherichia coli JM109, Escherichia coli BL21, RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli Dα, E. coli W3110, Bacillus subtilis (Bacillus subtilis), Bacillus Chuo ringen cis Bacillus strain, Salmonella typhimurium (Salmonella typhimurium), such as (Bacillus thuringiensis), Serratia marcescens and various enterococci such as Pseudomonas species and strains.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells and animal cells (for example, HEK (Human embryonic kidney) 293, CHO (Chinese hamster ovary) , W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines) and plant cells.

The present invention also provides a fused protein produced by the host cell, wherein the C-terminal domain of the VLRB protein derived from the feline eel-derived variable lymphocyte receptor B protein and the feline-derived VLRB protein from which the hydrophobic tail domain is removed.

The fusion protein according to the present invention is a fusion protein in which the C-terminal domain of the VLRB protein derived from the seventh eel is linked to the C-terminus of the stalk domain of the wild-type VLRB protein.

The present invention also provides a multivalent antibody having increased binding affinity to a target antigen, characterized in that the fusion protein is made of a multimer of 8-mer or 10-mer by self-assembly. The multivalent antibody of the present invention is a multimeric form of the Eagle VLRB fusion protein in which the hydrophobic tail domain at the C-terminus is replaced with the C-terminal sequence of the luciferous eel while maintaining the binding ability to the target antigen , And C-terminal domain of cucurbit eel rich in cysteine residues, such as 8-mer or 10-mer.

The term multimer, complex or polymer as used herein may be used interchangeably.

The polyvalent antibody of the present invention is a polyvalent antibody having 8 or 10 VLRB proteins, which is a wild-type antibody, and the antigen binding ability is remarkably increased.

The present invention also relates to

(a) transforming a host cell with the recombinant expression vector of the present invention;

(b) culturing the transformed host cell of step (a); And

(c) obtaining a multivalent antibody having increased binding ability to a target antigen, comprising the steps of: (a) obtaining a multimer of a fusion protein or a multimer of the fusion protein from the cultured host cell or culture thereof; And a manufacturing method thereof.

Specifically, the method for producing the multivalent antibody of the present invention comprises immunizing a mouse with an immunogen or an antigen, isolating lymphocytes by collecting the blood of the immunized eel, extracting the total RNA of the lymphocytes, and extracting mature VLRBs After securing mRNA, a PCR pool of VLRBs except a hydrophobic region at the terminal of carboxy was prepared by performing a polymerase chain reaction with the template as a template. Each of the clones of the prepared cDNA was cloned into a pTEL vector (Fig. 1B) containing the C-terminal base sequence of the luciferase eel VLRB. The C-terminal ends of the Eel VLRBs having different antigen recognition sequences and the VLRB A recombinant expression vector capable of expressing the fusion protein was prepared. Subsequently, 293-F cells were transfected with each recombinant expression vector, and transfected 293-F cells were cultured. After culturing, the reactivity with immunogen or antigen was confirmed by ELISA. Finally, clones with excellent binding ability to immunogens or antigens were selected and Western blotting of the culture of the selected clones under non-reducing and reducing conditions confirmed that the fusion protein was formed as a multimer. The immunogen or antigen may be, but is not limited to, a virus, a microorganism, and the like.

The present invention also provides an increased antibody binding to the target antigen produced by the method.

The multivalent antibody of the present invention has a multimerized form of the fusion protein in which the hydrophobic tail domain is replaced with the C-terminal sequence of the luciferase VLRB protein in the Eagle VLRB protein while retaining the binding ability to the target antigen. , And 8 or 10 horsetail VLRB proteins capable of binding to the target antigen. Thus, the antibody is a polyvalent antibody having a significantly increased antigen binding ability as compared to the monoclonal antibody.

The present invention also provides a method for detecting a target antigen by treating the suspected sample containing the polyvalent antibody with a target antigen.

In the target antigen detection method of the present invention, the antigen is not limited thereto, but may be a virus or a microorganism.

In the target antigen detection method of the present invention, the sample may be, but not limited to, a food, a water, a solution containing specific or unspecific microorganisms, a tissue, a cell, blood, serum, plasma, saliva and the like.

The target antigen detection method of the present invention can be carried out by an antigen-antibody reaction method. In this case, the multivalent antibody of the present invention which specifically binds to the target antigen to be detected can be used. The present invention can be carried out according to a conventional immunoassay method and used to detect the presence or absence of a target antigen. Such immunoassay can be carried out according to various quantitative or qualitative immunoassay methods developed in the past.

Detection of the conjugate of the multivalent antibody of the present invention and the target antigen can be performed by an indirect enzyme linked immunosorbent assay (ELISA) or a sandwich ELISA method, but is not limited thereto. Determination of the activity or signal of the final enzyme in the indirect ELISA method and the sandwich-ELISA method can be carried out according to various methods known in the art. Detection of such a signal enables qualitative or quantitative analysis of the polyvalent antibody of the present invention.

The present invention also provides a composition for detecting a target antigen containing the polyvalent antibody as an active ingredient, and a kit for detecting a target antigen comprising the composition as an effective ingredient.

The composition for detecting target antigens of the present invention contains a polyvalent antibody having a multimerized form of the Eagle's VLRB fusion protein in which the hydrophobic tail domain is replaced with that derived from the luciferous eel while the binding ability to the target antigen is maintained, The kit for immunoassay is preferably a kit for immunoassay, including but not limited to direct-ELISA, indirect-ELISA, sandwich-ELISA, protein microarray, radioimmunoassay (RIA: Radioimmunoassay) or the like.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

EXAMPLES Example 1: Secretion efficiency of wild-type Echinoids VLRB protein to the extracellular space

It was confirmed that the C-terminus of the hagfish VLRB protein is composed of amino acids with high hydrophobicity (FIG. 2A). The complete VLRB genes were amplified by PCR from the cDNA synthesized from the ejaculate blood mRNA and cloned into the pKINGe / ccdB vector. Among them, randomly selected plasmids were transfected into 293-F cell lines to express VLRB proteins. Two days after transduction, supernatants and pellets of cells were separated and recovered separately. The cell culture was filtered with a 0.45 ㎛ membrane filter to remove debris and concentrated 10 times with a freeze dryer. The recovered cells were treated with a cell lysis solution containing a protease inhibitor cocktail (Promega, USA) to induce the elution of proteins in the cells. Cell cultures and cell samples prepared as described above were electrophoresed in a loading buffer containing 1% β-mercaptoethanol (hereinafter, 2-ME). After electrophoresis, the protein was transferred to a PVDF membrane, and then the VLRB protein was identified using a mouse anti-VLRB antibody 11G5 capable of detecting the iguer VLRB protein. As a result, the recombinant VLRB protein was observed in the cell pellet in all of the selected eight clones, but the VLRB protein was confirmed in some clones in the cell culture (FIG. 2B). The above results indicated that the efficiency of the wild-type Eagle VLRB protein secreted outside the cell was low.

Example 2 C-terminal Sequence Substitution of Egg VLRB

In the present invention, the C-terminal sequence of the VLRB is replaced with the C-terminal sequence of the VLRB of the VLRB to overcome the low secretion efficiency of the wild-type VLRB protein to the outside of the cell and induces complex formation to improve the binding ability of the VLRB antibody to the antigen Respectively.

First, in order to establish a recombinant protein expression system through human cell lines, a cell line derived from a human embryonic kidney (HEK), which is known to have the highest expression rate of foreign genes, can be grown as a suspension, 293-F cell line was selected and used. For the development of a vector system capable of expressing in a human cell line, the inventors constructed a recombinant expression vector using pTracer-EF / V5-His (# V887-20, Invitrogen, USA) as a basic framework. First, secretory signal peptides were introduced for the isolation of exogenous proteins into the extracellular space. To this end, pSecTag2A (# V900-20, Invitrogen) murine (murine) Ig κ chain leader sequence for the polymerase chain reaction from the vector (polymerase chain reaction, PCR) to amplify and, VLRB Sfi I restriction to facilitate gene transfer the enzyme seat is connected to pull air to remove the Cm / ccdB gene, located at both ends from the vector pEF-DEST gateway (# 12285-011, Invitrogen) and cloned between the pTracer-EF / V5-His vector of the Kpn I and Not I pKINGeo / ccdB vector (FIG. 1A).

Afterwards, the LC sequence was fused to the stalk part of the hagfish VLRB gene randomly selected by the PCR technique to replace the C-terminal sequence of the horsetail VLRB with the C-terminal (lamprey C-term, hereinafter referred to as LC) (Fig. 3A).

Primer set for C-terminal fusion of Chilo suppressor VLRB Primer name The base sequence (5 '- > 3') SEQ ID NO: Primer SP CAGGTACCATGGAGACAGACACACTCCTG 3 Primer 1 TTGTGCAGGCGGGCTTTCCGCAGTCGCCACCGTCCGCGCACGCGTTCATGACACG 4 Primer 2 CGGAAAGCCCGCCTGCACAACTCTCCTGAACTGCGCGAATTTCCTCAGCTGCCT 5 Primer 3 TCAACGTTTCCTGCAGAGGGCGCAGGTCGAGCAGAGGCAGCTGAGGAAATTCGC 6

The PCR product was cloned into pKINGeo / ccdB vector and transfected with 293-F cell line to confirm the expression pattern. As a result, it was confirmed that the VLRB recombinant protein of LC-Fusiformis VLRB was detected in both the cell culture medium and the cell lysate (Fig. 3B). In particular, the amount of VLRB protein secreted to the outside of the cell was found to be larger than that of the wild-type VLRB. In addition, VLRB of several types of complex forms (bidentate, quaternary, octamer, and octamer), including monomer size, was confirmed in the results of electrophoresis under nonreducing conditions.

Example 3. Expression Tendency Analysis of Eagle VLRB Fusion Protein Substituted with C-terminal Sequence of Seven Eel

The LC sequence was used to construct a pTEL vector containing the LC sequence in order to produce the lyophilized VLRB proteins in the form of a library. The pTEL vector was constructed in such a manner that the cloned VLRB gene was expressed as an LC sequence by introducing an LC sequence in place of the existing V5 and 6 x His tag sequences using the pKINGeo / ccdB vector as a framework (Fig. 1B ).

Total RNA was extracted from the lymphocytes in the blood of broiler chicks and library-type VLRBs cDNA was obtained as a template. The obtained VLRB gene was cloned into pTEL vector and transformed into E. coli DH5? Competent cells. The library was verified by identifying VLRB insertion genes of various sizes from randomly selected colonies (FIG. 4B). Randomly selected VLRB plasmids were transfected with 293-F cell lines, respectively, and expression patterns were confirmed by Western blotting. Three days after transduction, cell cultures were collected and subjected to electrophoresis under non-reducing conditions without addition of 2-ME. As a result, the expression of VLRB protein was confirmed in the remaining 11 clones except 3 clones (3LC, 6LC, 14LC) (Fig. 4C). Especially, all of the clones expressing the LC fusion VLRB protein exhibited more than two diploid complexes, and it was confirmed that all of the clones expressing the LC fusion VLRB protein contained complexes of 8 or 10 diploids although the degree of expression was different. Based on the above results, the present inventors confirmed that the LC fusion VLRB proteins produced in the pTEL vector secrete VLRB protein in a high proportion (78.6%) of the cell culture medium, and most of them form a complex form.

Example 4 Screening of Fusion Proteins Having Specific Antibody-Specific Binding Ability

Viral hemorrhagic Septicemia virus (VHSV), the cause of viral hemorrhagic septicemia (VHS), is known to cause damage mainly to cultured flounder in winter and spring water temperatures in Korea. The present inventors obtained the VLRB library from the horses immunized with VHSV and cloned it as a pTEL vector in order to prepare a hatching VLRB antibody for VHSV. The recombinant plasmids were transformed into E. coli DH5? Competent cells, amplified, and transfected with 293-F cell lines, respectively. ELISA was performed using cell culture 3 days after transduction in order to find a VLRB antibody specific for VHSV.

Experiments were performed by treating multiwell plates coated with VHSV (200 ng) or HEL (20 ng) antigen with 3% skim milk solution and transfected 293-F cell culture supernatants. Two clones, 43LC and 7LC, which showed the highest reactivity to VHSV among a total of 96 clones, were selected. For the further experiments of 7LC clone with nonspecific binding ability in both VHSV and HEL and 43LC showing specific reactivity in VHSV, two streaking steps were performed and DNA of high purity was extracted with DNA prep kit. We analyzed the amino acid sequences of these two clones and analyzed the expression patterns from 293-F cell lines via Western blotting.

Sequence analysis results of fusion proteins 43LC and 7LC 43LC 7LC LRR1 VLWLGGNKIPSLPHGVFD (SEQ ID NO: 7) VLQLQGNKLQSLPSGVFD (SEQ ID NO: 14) LRRv1 KLTSLTLLSLHTNQLQSLPDGVFD (SEQ ID NO: 8) KLTQLTYLSLSTNQLQSLPNGVFD (SEQ ID NO: 15) LRRv2 KLTSLTLLSLHTNQLQSLPSGVFD (SEQ ID NO: 9) KLTQLTVLGLQTNQLKSVPDGVFD (SEQ ID NO: 16) LRRv3 KLTELKELRLYENKLQSLPHGVFD (SEQ ID NO: 10) - LRRv4 KLTQQLKDLRLHQNQLKSVPDGVFD (SEQ ID NO: 11) - CP RLTSLQTIYLYSNP (SEQ ID NO: 12) RLTSLQKIYLYSNP (SEQ ID NO: 17) LRRCT WDCTCPGVDYLSRWLHTNSKKETSDSAKCSGSGFPVRSIICP (SEQ ID NO: 13) WDCTCPGIRYFSEWINKHSGVVRDSSNNVNPDSAKCSGSGKPVRSIICP (SEQ ID NO: 18)

These two clones differed significantly in the LRRv module, which plays an important role in recognizing antigens. Compared to the 43LC containing a total of 4 LRRv modules, 7LC has two LRRv modules (Table 2). In addition, the molecular weight of 43LC recombined by the difference in the number of modules was confirmed to be larger than 7LC by western blotting (Fig. 6A).

Since the two selected clones showed nonspecific binding ability in ELISA, their antigen specificity was examined using various antigens. As a result of testing the reactivity of 43LC and 7LC clones against wells coated with VHSV, AIV (avian influenza virus), HA (hemagglutinin) and HEL antigens and wells not coated with antigens (AgX) It was identified as a non-specific binding ability clone with high OD value irrespective of kinds. On the other hand, 43LC showed a slight OD value but showed the highest binding ability to VHSV and antigen specific reactivity (FIG. 6B). In order to eliminate the nonspecific reactivity observed above, 43LC and 7LC were treated with 5% skim milk solution at a ratio of 1: 1 and then subjected to ELISA. As a result, the nonspecific binding of the 43LC clone having the specific binding ability to VHSV was confirmed to be lower, and the 7LC clone showed almost no reactivity with all the antigens (FIG. 6C). The binding ability of 43LC to VHSV antigen and VHSV surface glycoprotein (G protein) was confirmed by western blotting to identify sites of VHSV antigen recognized by 43LC. Briefly, each antigen was transferred to a PVDF membrane, treated with a primary antibody for each of the 43LC and 7LC clones, and treated with a secondary antibody for anti-mouse antibody (11G) capable of recognizing the stalk region of the avian VLRB. As a result, no reaction was observed in the membrane treated with the primary antibody of 7LC, whereas the VHSV and the recombinant G protein were confirmed in the membrane treated with the primary antibody of 43LC (FIG. 6D). From the above results, it was found that the 43LC clone specifically binds to the G protein, an outer membrane protein of the VHS virus.

In addition, the HINAE (Hirame natural embryo cell) cell line derived from flounder was infected with VHSV at a concentration of 1 × 10 ⁵ TCID ⁵⁰ / ml and the binding ability of the 43LC clone to the amplified VHS virus was confirmed by immunocytochemistry. As a result, as shown in FIG. 7, 43LC was specifically bound to the VHS virus and green fluorescence was confirmed. No fluorescence was observed in cells treated with 7LC in accordance with the above results.

Example 5: Antigen binding ability of monomers and polymers of fusion proteins having specific binding ability of target antigen

The formation of polymers with or without the presence of LC sites leading to polymer formation of the 43LC clone was investigated. As shown in FIG. 8, the 43LC containing the LC region and the mono 43 protein, which genetically deleted the LC region, were identified as complexes and monomers in the non-reducing condition without 2-ME treatment, And all of them were separated into a single form under reducing conditions.

The VHSV-specific binding potency of these two antibodies, mono-43 in the form of monomers 43 and 43LC in the polymer form, was investigated by ELISA. As a result, a high specific binding capacity to VHSV was observed only in the polymer 43LC (FIG.

The wild-type VLRB antibody in which the hydrophobic tail domain of the present invention was substituted with the C-terminal sequence of the VLRB was formed as a complex upon expression in the transduced cells and exhibited increased extracellular secretion ability against the wild type VLRB, RTI ID = 0.0 > monoclonal < / RTI > sequence. VLRB antibodies selected by the method of the present invention may be used for various antibody-related experiments such as mouse antibodies.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY          IMMUCELL CO., LTD. <120> Fusion protein comprising C-terminus from lamprey VLRB protein          linked to hagfish VLRB protein with deleted hydrophobic tail          domain and uses thereof <130> PN17036 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 63 <212> DNA <213> Mus musculus <400> 1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gac 63 <210> 2 <211> 102 <212> DNA <213> Lampetra japonica <400> 2 gacggtggcg actgcggaaa gcccgcctgc acaactctcc tgaactgcgc gaatttcctc 60 agctgcctct gctcgacctg cgccctctgc aggaaacgtt ga 102 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caggtaccat ggagacagac acactcctg 29 <210> 4 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttgtgcaggc gggctttccg cagtcgccac cgtccgcgca cgcgttcatg acacg 55 <210> 5 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cggaaagccc gcctgcacaa ctctcctgaa ctgcgcgaat ttcctcagct gcct 54 <210> 6 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tcaacgtttc ctgcagaggg cgcaggtcga gcagaggcag ctgaggaaat tcgc 54 <210> 7 <211> 18 <212> PRT <213> Eptatretus burgeri <400> 7 Val Leu Trp Leu Gly Gly Asn Lys Ile Pro Ser Leu Pro His Gly Val   1 5 10 15 Phe Asp         <210> 8 <211> 24 <212> PRT <213> Eptatretus burgeri <400> 8 Lys Leu Thr Ser Leu Thr Leu Leu Ser Leu His Thr Asn Gln Leu Gln   1 5 10 15 Ser Leu Pro Asp Gly Val Phe Asp              20 <210> 9 <211> 24 <212> PRT <213> Eptatretus burgeri <400> 9 Lys Leu Thr Ser Leu Thr Leu Leu Ser Leu His Thr Asn Gln Leu Gln   1 5 10 15 Ser Leu Pro Ser Gly Val Phe Asp              20 <210> 10 <211> 24 <212> PRT <213> Eptatretus burgeri <400> 10 Lys Leu Thr Glu Leu Lys Glu Leu Arg Leu Tyr Glu Asn Lys Leu Gln   1 5 10 15 Ser Leu Pro His Gly Val Phe Asp              20 <210> 11 <211> 25 <212> PRT <213> Eptatretus burgeri <400> 11 Lys Leu Thr Gln Gln Leu Lys Asp Leu Arg Leu His Gln Asn Gln Leu   1 5 10 15 Lys Ser Val Pro Asp Gly Val Phe Asp              20 25 <210> 12 <211> 14 <212> PRT <213> Eptatretus burgeri <400> 12 Arg Leu Thr Ser Leu Gln Thr Ile Tyr Leu Tyr Ser Asn Pro   1 5 10 <210> 13 <211> 42 <212> PRT <213> Eptatretus burgeri <400> 13 Trp Asp Cys Thr Cys Pro Gly Val Asp Tyr Leu Ser Arg Trp Leu His   1 5 10 15 Thr Asn Ser Lys Lys Glu Thr Ser Asp Ser Ala Lys Cys Ser Gly Ser              20 25 30 Gly Phe Pro Val Arg Ser Ile Ile Cys Pro          35 40 <210> 14 <211> 18 <212> PRT <213> Eptatretus burgeri <400> 14 Val Leu Gln Leu Gln Gly Asn Lys Leu Gln Ser Leu Pro Ser Gly Val   1 5 10 15 Phe Asp         <210> 15 <211> 24 <212> PRT <213> Eptatretus burgeri <400> 15 Lys Leu Thr Gln Leu Thr Tyr Leu Ser Leu Ser Thr Asn Gln Leu Gln   1 5 10 15 Ser Leu Pro Asn Gly Val Phe Asp              20 <210> 16 <211> 24 <212> PRT <213> Eptatretus burgeri <400> 16 Lys Leu Thr Gln Leu Thr Val Leu Gly Leu Gln Thr Asn Gln Leu Lys   1 5 10 15 Ser Val Pro Asp Gly Val Phe Asp              20 <210> 17 <211> 14 <212> PRT <213> Eptatretus burgeri <400> 17 Arg Leu Thr Ser Leu Gln Lys Ile Tyr Leu Tyr Ser Asn Pro   1 5 10 <210> 18 <211> 49 <212> PRT <213> Eptatretus burgeri <400> 18 Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Phe Ser Glu Trp Ile Asn   1 5 10 15 Lys His Ser Gly Val Val Arg Asp Ser Ser Asn Asn Val Asn Pro Asp              20 25 30 Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Val Arg Ser Ile Cys          35 40 45 Pro    

Claims (10)

A recombinant expression vector comprising a polynucleotide encoding a C-terminal domain of a VLRB protein derived from a lentil-eel-derived &lt; RTI ID = 0.0 &gt; Chlamydophilus &lt; / RTI &gt; linked to the 3'-end of a gene encoding a horseshoe-derived variable lymphocyte receptor B (VLRB) protein from which the hydrophobic tail domain has been removed. 2. The method of claim 1, wherein the VRRB protein from which the hydrophobic tail domain is removed comprises a signal peptide (SP), an N-terminal capped LRR, a leucine-rich repeat (LRR) (variable LRR modules), a connecting peptide (CP), a C-terminal capped LRR (LRRC), and a Stalk domain. [Claim 3] The recombinant expression vector according to claim 2, wherein the VLRB protein from which the hydrophobic tail domain is removed is a murine immunoglobulin kappa chain leader sequence of the signal peptide (SP). A host cell transformed with the recombinant expression vector of claim 1. A fusion protein produced by the host cell of claim 4, wherein the C-terminal domain of the VLRB protein derived from the wild-type eel-derived variable lymphocyte receptor B (VLRB) protein is removed. A multivalent antibody having increased binding capacity to a target antigen, characterized in that the fusion protein of claim 5 consists of a multimer of 8-mer or 10-mer by self-assembly. (a) transforming a host cell with the recombinant expression vector of claim 1;
(b) culturing the transformed host cell of step (a); And
(c) obtaining a multivalent antibody having increased binding ability to a target antigen, comprising the steps of: (a) obtaining a multimer of a fusion protein or a multimer of the fusion protein from the cultured host cell or culture thereof; Gt; 17. A multivalent antibody having increased binding capacity to a target antigen produced by the method of claim 7. 8. A method for detecting a target antigen by treating a multivalent antibody of claim 6 or 8 to a suspected sample containing a target antigen. A composition for detecting a target antigen, which comprises the polyvalent antibody of claim 6 or 8 as an active ingredient.

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